Central to the development of human oral biofilm communities are microbial interactions that drive the spatial arrangement within bacterial communities. Such communities on enamel form supragingival dental plaque. These intimate interactions are facilitated by physical interactions called coaggregations, which are specific adherences of genetically distinct partner cells that bind to one another to form multicellular networks such as the multispecies communities of human dental plaque. Interactions among oral streptococci and actinomyces dominate initial dental plaque development. In this reporting period, we used a DNA microarray to identify Streptococcus gordonii genes regulated in response to coaggregation with Actinomyces naeslundii. Expression of 23 genes changed >3-fold in coaggregates, including nine genes involved in arginine biosynthesis and transport. The capacity of S. gordonii to synthesize arginine was assessed using a chemically defined growth medium. In monoculture, streptococcal arginine biosynthesis was inefficient and streptococci could not grow aerobically in low arginine. In dual-species cultures containing coaggregates, however, S. gordonii grew to high cell density in low arginine. Equivalent co-cultures without coaggregates showed no growth until coaggregation was evident, which occurred after 9 h of incubation. An argH mutant was unable to grow in low arginine with or without A. naeslundii, indicating that arginine biosynthesis was essential for coaggregation-induced streptococcal growth. Using quantitative RT-PCR, expression of argC, argG and pyrAb was strongly (10- to 100-fold) up-regulated in S. gordonii monocultures after 3 h growth when exogenous arginine was depleted. Co-cultures without induced coaggregation showed similar regulation. However, within 1 h after coaggregation with A. naeslundii, expression of argC, argG and pyrAb in S. gordonii was partially up-regulated although arginine was plentiful, and mRNA levels did not increase further when arginine was diminished. Thus, A. naeslundii stabilizes S. gordonii expression of arginine biosynthesis genes in coaggregates and enables aerobic growth when exogenous arginine is limited. Metabolic cooperation among bacteria may be important to the repetitive and distinctive community composition of initial oral biofilm communities, and food webs could be set up through this cooperation. The mechanisms of communication among mixed-species coaggregates is a topic of much interest in my laboratory.[unreadable] [unreadable] The initial colonizers of tooth surfaces are a specific subset of the oral microflora. Of these bacteria, those that colonize the clean enamel surface independently of other bacteria possess mechanisms for attachment to the acquired salivary pellicle covering the enamel and possess the ability to metabolize salivary components as the sole nutritional source. We modelled the in vivo environment by using a flow device and sorbarod filters (paper-wrapped sheaf of tightly packed fibers: cylinder 10 mm diameter; 20 mm length) with saliva for nutrition. High cell densities were achieved, and we evaluated the ability of a streptococcus-actinomyces community to produce the universal signaling molecule autoinducer-2 (AI-2). Oral commensal bacteria Streptococcus oralis 34 and Actinomyces naeslundii T14V were grown as single-species and dual-species biofilms. After 48 h, dual-species biofilm communities of interdigitated S. oralis 34 and A. naeslundii T14V contained 3.2x109 cells: 5-fold more than single-species biofilms. However, these 48-h dual-species biofilms exhibited the lowest AI-2 concentration ratio (AI-2 concentration as nanomoles/L in the biofilm to cell density as cell number/mL of the biofilm). The more than 10-fold decrease in concentration ratio seen between 1-h and 48-h S. oralis 34-A. naeslundii T14V biofilms suggests that peak production of AI-2 occurs early in community development and is followed by a very low steady-state level. Specific concentrations of AI-2 appear to be essential for the initiation of oral commensal biofilm communities. Our long-range goal is to understand the molecular mechanisms of cellular communication and their relationship to the spatiotemporal development and establishment of dental plaque.